Several hundred naturally occurring, monoterpenes are known, and essentially all are biosynthesized from geranyl pyrophosphate, the ubiquitous C.sub.10 intermediate of the isoprenoid pathway (Croteau and Cane, Methods of Enzymology 110:383-405 [1985]; Croteau, Chem. Rev. 87:929-954 [1987]). Monoterpene synthases, often referred to as "cyclases," catalyze the reactions by which geranyl pyrophosphate is cyclized to the various monoterpene carbon skeletons. Many of the resulting carbon skeletons undergo subsequent oxygenation by cytochrome P450 hydroxylases to give rise to large families of derivatives. Research on biosynthesis has been stimulated by the commercial significance of the essential oils (Guenther, The Essential Oils, Vols. III-VI (reprinted) R. E. Krieger, Huntington, N.Y. [1972]) and aromatic resins (Zinkel and Russell, Naval Stores: Production, Chemistry, Utilization, Pulp Chemicals Association, New York [1989]) and by the ecological roles of these terpenoid secretions, especially in plant defense (Gershenzon and Croteau, in "Herbivores: Their Interactions with Secondary Plant Metabolites," Vol. I, 2nd Ed. (Rosenthal and Berenbaum, eds.) Academic Press, San Diego, Calif., pp. 165-219 [1991]; Harborne, in "Ecological Chemistry and Biochemistry of Plant Terpenoids," (Harborne and Tomas-Barberan eds.) Clarendon Press, Oxford, Mass., pp. 399-426 [1991]).
The reactions catalyzed by the cytochrome P450-(-)-limonene hydroxylases determine the oxidation pattern of the monoterpenes derived from limonene (see FIGS. 1A-1C). These reactions are completely regiospecific and are highly selective for (-)-limonene as substrate. The primary products of limonene hydroxylation (trans-carveol and trans-isopiperitenol) are important essential oil components and serve as precursors of numerous other monoterpenes of flavor or aroma significance (see FIGS. 1A-1C).
One of the major classes of plant monoterpenes is the monocyclic p-menthane (1 -methyl-4-isopropylcyclohexane) type, found in abundance in members of the mint (Mentha) family. The biosynthesis of p-menthane monoterpenes in Mentha species, including the characteristic components of the essential oil of peppermint (i.e., (-)-menthol) and the essential oil of spearmint (i.e., (-)-carvone), proceeds from geranyl pyrophosphate via the cyclic olefin (-)-limonene and is followed by a series of enzymatic redox reactions that are initiated by cytochrome P450 limonene hydroxylases (e.g., limonene-3-hydroxylase in peppermint and limonene-6-hydroxylase in spearmint and related species; Karp et al., Arch. Biochem. Biophys. 276:219-226 [1990]; Gershenzon et al., Rec. Adv. Phytochem. 28:193-229 [1994]; Lupien et al., Drug Metab. Drug Interact. 12:245-260 [1995]). The products of limonene hydroxylation and their subsequent metabolites (as shown in FIGS. 1A-1C) also serve ecological roles in plant defense mechanisms against herbivores and pathogens, and may act as signals in other plant-insect relationships (e.g., as attractants for pollinators and seed dispersers).
A detailed understanding of the control of monoterpene biosynthesis and of the reaction mechanisms, enzymes and the relevant cDNA clones as tools for evaluating patterns of developmental and environmental regulation, for examining active site structure-function relationships and for the generation of transgenic organisms bearing such genes are disclosed in part in parent U.S. related application Ser. No. 08/582,802 filed Jan. 4, 1996 as a continuation of application Ser. No. 08/145,941 filed Oct. 28, 1993, the disclosures of which are incorporated herein by this reference, which disclose the isolation and sequencing of cDNAs encoding (-)4S-limonene synthase, the enzyme responsible for cyclizing geranyl pyrophosphate to obtain (-)-limonene. To date, however, no information has been available in the art regarding the protein and nucleotide sequences relating to the enzymes through which (-)-limonene is hydroxylated (by the action of (-)-limonene-6-hydroxylase to form trans-carveol or by the action of (-)-limonene-3-hydroxylase to form trans-isopiperitenol as shown in FIG. 1).